Zinc is a pivotal ion in the cellular economy as an enzyme cofactor and a structural element in proteins. It has been known for some time that specific populations of glutamatergic neurons in the mammalian forebrain have high levels of chelatable-zinc in their synaptic vesicles. The precise role of this vesicular-Zn remains enigmatic, as does the reason for the division of the nervous system into Zn-rich and Zn-poor glutamatergic terminals. For some time Zn has been thought to be released during the course of synaptic transmission and act as a neuromodulator. We have shown recently that little Zn (-8 pM) is released during neurotransmission making its role as a neuromodulator questionable, reopening the debate on the function of vesicular Zn. The long-range goal of this proposal is to understand the role that transition metals play in synaptic transmission in the mammalian CNS. The objective of this application is to provide an understanding of the role of free-Zn in synaptic vesicles. Our central working hypothesis is that vesicular-Zn serves both a role in signal transduction within synaptic vesicles and tethered to proteins on the extracellular face of the presynaptic membrane. However, under conditions of profound stress Zn may be released and serve as a neurotoxic agent. Once the normal role of Zn has been established it will serve as a starting point for intervening in the numerous pathological situations where Zn has been implicated. We are particularly well prepared to undertake this research because we have developed fluorometric probes, specific Zn chelators and methods that will allow us to detect Zn in different cellular compartments in the brain. We plan to test our central hypothesis and accomplish the overall objective of this application by pursuing the following three specific aims: Aim 1: To determine the source and function of the extracellular chelatable-Zn that is found in certain sectors of the hippocampus that we have termed the "Zn-veneer." Zn-sensitive fluorescent probes and inductively coupled plasma mass spectroscopy (ICPMs), in combination with transgenic mice that lack vesicular Zn will be used to accomplish this aim. We hypothesize that under normal circumstances Zn is not released into the extracellular space but remains bound to sites on the plasma membrane. We conjecture that the Zn on the veneer can bind certain molecules in the extracellular space forming ternary complexes that may play a role in signal transduction. A fluorescent molecule designed to form ternary complexes will be used to test this hypothesis. Aim 2: To determine the mechanism whereby Zn is transported into cells in the hippocampus. We will use fluorescent Zn-probes in hippocampal slices in order to determine how Zn enters neurons from the extracellular space. Aim 3: To determine the source of Zn that appears in neurons after a period of ischemia. We will use an in vitro model of ischemia based on the oxygen and glucose deprivation of hippocampal slices. The release of Zn will be measured both with ICPMs and fluorometric probes. Experiments will be performed to determine the origin of the Zn, its mechanism of release and its site of subcellular accumulation after ischemia. The proposed research is innovative because it capitalizes on novel imaging techniques that use unique reagents that were developed in our laboratory. Our expectations are that, at the conclusion of this project, we will have provided a detailed view of the mechanisms that control the passage of Zn between cellular compartments in neurons. Unregulated free Zn ions have been implicated in a number of neuropathologies, among these, stroke, Alzheimer's disease and epilepsy. A thorough understanding of the normal mechanisms of Zn homeostasis and how such mechanisms are altered in neuropathologies will be essential in the development of effective strategies for treatment